solid™ system s o d - ndsumcclean/plsc411/solid... · solid system: development status dedicated...

SOLiD™ SystemSequencing by Oligonucleotide Ligation and Detection

2 November 2006 © 2006 Applied Biosystems

Workflow

Application specificsample

preparation

Application specificsample

preparation

Emulsion PCR &

substrate preparation

Emulsion PCR &

substrate preparation

Imaging &basecallingImaging &

basecalling

Application specific

Data analysis

Application specific

Data analysis

Sequencing chemistry

Sequencing chemistry

3 November 2006 © 2006 Applied Biosystems

Fragment Library (directed resequencing)

Mate Pair Library (whole genome sequencing)

DNA Fragment

60-90 Bases

Create library of DNA fragments:2 methods

1µmbead

P1 Adapter P2 Adapter

1µmbead

P1 Adapter Internal Adapter

25 baseTag #1

25 baseTag #2

P2 Adapter

4 November 2006 © 2006 Applied Biosystems

Creating a mate-paired library (i)

Randomly Fragmentsample

Complex sample

Ligate Internal adapters

Circularize

+

size select(eg:1, 2, 3, 5, 10 KB)

5 November 2006 © 2006 Applied Biosystems

Creating a mate-paired library (ii)

Cleave EcoP15I

27bp 27bp

Ligate P1 and P2

+

Internal Adapter

25 baseTag #1

25 baseTag #2

P1 P2

6 November 2006 © 2006 Applied Biosystems

Emulsion PCR (i)

+

Templates

Primers P1<<P2

Enzyme

P1-coupled beads

Polymerase

7 November 2006 © 2006 Applied Biosystems

Emulsion PCR (ii)Mix PCR aqueous phase into a water-in-oil (w/o) emulsion and carry out emulsion PCR

8 November 2006 © 2006 Applied Biosystems

Emulsion MetricsBead size: 1 μm

Reactor size: 4 μmReactor volume: 34 fL

Beads / emulsion plate (96-well): 2-4 x 109

Post Enrichment: ~500M / plate

SOLiD system: Clonal Amplification

Dressman, PNAS 2003Embleton, 1994

9 November 2006 © 2006 Applied Biosystems

emulsion PCR Individual Bead

2) Polymerase extends from P1

1) Template Anneals to P1

3) Complementary sequence is extended off bead surface

4) Template disassociates

10 November 2006 © 2006 Applied Biosystems

Results of emulsion PCR,after breaking emulsion

Bead contains ~20K amplified products from original single strand molecule Beads with no product

11 November 2006 © 2006 Applied Biosystems

Enrichment

P1 P2

P2’

P2 P1

LargePolystyrenebead coated

with P2

Centrifuge in glycerol gradient

Supernatant Captured beads with templates

Pellet Beads with no template

12 November 2006 © 2006 Applied Biosystems

P2-hybridization, Pre- and post-enrichment

Pre-enrichment (30%)

Post-enrichment (80%)

em6395

13 November 2006 © 2006 Applied Biosystems

Bead Deposition

3’-end modification

Beads attached to glass surface in a random array

14 November 2006 © 2006 Applied Biosystems

Properties of the ProbesSpatial separation among dye, ligation & cleavage sites

3’

n n n T C z z z

Fluorescent dye

Cleavage site,

3’ Ligation site

1,024 Octamer Probes (45)4 Dyes, 4 dinucleotides, 256 probes per dyeN= degenerate bases Z= Universal bases

15 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligationLigation reaction

ligase3’ p5’

universal seq primer

Template Sequence 5’ 3’P1 Adapter

1µmbead

5’

3’ 5’

3’ 5’

3’ 5’

n n n T A z z z

n n n T C z z z n n n A T z z z

n n n G G z z z

1µmbead

universal seq primer p5’

16 November 2006 © 2006 Applied Biosystems

ligase

SOLiD Chemistry System 4-color ligation Ligation reaction

ligase3’ p5’

universal seq primer

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

p5’T AT A

5’

3’ 5’

3’ 5’

3’ 5’

n n n T A z z z

n n n T C z z z n n n A T z z z

n n n G G z z z

17 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligation De-Phosphorylation

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

Template Sequence 5’ 3’P1 Primer

1µmbead

universal seq primer

1µmbead

p5’

T AT A

18 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligation Visualization

4,5 Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

T AT A

19 November 2006 © 2006 Applied Biosystems

T AT A

SOLiD Chemistry System 4-color ligationCleavage

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

p5’

4,5

20 November 2006 © 2006 Applied Biosystems

ligase

SOLiD Chemistry System 4-color ligation Ligation (2nd cycle)

ligase

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

p5’

5’

3’ 5’ 3’ 5’

n n n T A z z z

G G

n n n T C z z z n n n A T z z z

3’ 5’n n n G G z z z

4,5

T AT A

21 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligation Visualization (2nd cycle)

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

G G

9,104,5

T AT A

22 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligation Cleavage (2nd cycle)

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

G GT AT A

9,104,5

p5’

23 November 2006 © 2006 Applied Biosystems

C CT TG AG A

19,2014,15

SOLiD Chemistry System 4-color ligationinterrogates every 5th base

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer

1µmbead

G GT AT A

9,104,5 24,25

24 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligation Reset

Template Sequence 5’ 3’P1 Adapter

1µmbead

1µmbead

25 November 2006 © 2006 Applied Biosystems

ligase

SOLiD Chemistry System 4-color ligation (1st cycle after reset)

ligase3’ p5’

universal seq primer n-1

Template Sequence 5’ 3’P1 Adapter

1µmbead

1µmbead

universal seq primer n-1 p5’

5’

3’ 5’

3’ 5’

3’ 5’

n n n T A z z z

n n n T C z z z n n n A T z z z

n n n G G z z z

A T

26 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligation (1st cycle after reset)

Template Sequence 5’ 3’P1 Adapter

1µmbead

universal seq primer n-1 A T

1µmbead

3,4

27 November 2006 © 2006 Applied Biosystems

SOLiD Chemistry System 4-color ligation (2nd Round)

5’ 3’P1 Adapter

1µmbead

universal seq primer n-1

1µmbead

A T A GT T G A C G

3,4 8,9 13,14 18,19 23,24

28 November 2006 © 2006 Applied Biosystems

Sequential rounds of sequencingMultiple cycles per round

3’universal seq primer

4,5 9,10 14,15 19,20 24,25

Template Sequence 5’ 3’P1 Adapter

1µmbead

1µmbead

3’universal seq primer n-1 reset

3’universal seq primer n-2reset

3’universal seq primer n-3reset

3’universal seq primer n-4reset

3,4 8,9 13,14 18,29 23,24

2,3 7,8 12,13 17,18 22,23

1,2 6,7 12,13 16,17 21,22

0,1 5,6 11,12 15,16 20,21

29 November 2006 © 2006 Applied Biosystems

1µmbead

P1 Adapter Internal Adapter

25 baseTag #1

25 baseTag #2

P2 Adapter

Paired End two sequences generatedSequential rounds of sequencingMultiple cycles per round

30 November 2006 © 2006 Applied Biosystems

2 base encoding● Double base interrogation eases the discrimination

between system errors and true polymorphism

A C G G T C G T C G T G T G C G T

A C G G T C G T C G T G T G C G T

A C G TA

C

G

T

5th Base

4thB

ase

31 November 2006 © 2006 Applied Biosystems

Benefits of SOLiD Chemistry System● Terminating Chemistry

– No issues with homopolymers● Capping step eliminates dephasing

– Control increase in noise with subsequent cycles ● Primer reset

– removes accumulated out-of-phase ligations– longer reads (Signal recovers)

● Ligation– high fidelity– high read quality

● Double interrogation of each base– unique to ligation method– increases base calling accuracy– SNP results in a two-color change

32 November 2006 © 2006 Applied Biosystems

10-box Linux Cluster15 Tb data storage

Dual Flow CellReagent

handling

SOLiD System Instrumentation High speed automated bead imaging

33 November 2006 © 2006 Applied Biosystems

4-Color Overlay

1/6th

Full Array Small Array

1800 panels 300 panels~20,000 beads/panel

SOLiD System Arrays: 4 Mega pixel Camera

34 November 2006 © 2006 Applied Biosystems

Improved feature finding

Beads called per panel: 22,693 49,980

35 November 2006 © 2006 Applied Biosystems

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5(base 4) (base 9) (base 14) (base 19) (base 24)

Round 2 after Reset

Reset prevents excessive noise accumulation

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5(base 5) (base 10) (base 15) (base 20) (base 25)

Round 1

36 November 2006 © 2006 Applied Biosystems

SOLiD System: Development Status● Dedicated teams at Beverly and Foster City with Instruments up and running ● Anticipated Performance

– Read length: 25x2 mate pair – Raw data output rate: anticipate 100-500 Mb per day– Raw accuracy: ≥98%– Consensus accuracy: >99.99% (@ 20x coverage)– Quality metrics: Compatible with KB™ Basecaller Software– Cost per run: Competitive– Libraries: Mate pair or Fragment

○ as required by application– Application Support

○ Whole genome resequencing○ Directed (Medical) resequencing○ Gene expression○ Tag Counting

37 November 2006 © 2006 Applied Biosystems

● For Research Use Only. Not for use in diagnostic procedures.

● Applera, Applied Biosystems, AB (Design) are registered trademarks and KB and SOLiD are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.